FUNDAMENTALS OF MICROBIOLOGY - E. Y. Tyumentseva - 2015

CONCLUSION

Microorganisms began colonizing the Earth 3 to 4 billion years ago, long before The Emergence of Higher Plants and animals. Microbes represent the most numerous and diverse group of living organisms. They are exceptionally widespread in nature and remain the only form of living matter capable of inhabiting virtually any substrate or environment, including highly organized PLANT AND ANIMAL hosts.

Microorganisms are essential for driving major Industrial processes, such as baking, winemaking, and brewing, as well as the Production of organic acids, Enzymes, dietary Proteins, Hormones, Antibiotics, and other Pharmaceuticals.

Successfully addressing challenges related to improving the quality and Nutritional Value of food products requires a comprehensive system of measures aimed at optimizing both production sanitation and environmental conditions.

In Russia, state control over sanitary and production hygiene is enforced by public health authorities. Water and food safety is guaranteed through established standards and integrated testing Methods, including sanitary microbiological analyses.

It is impossible to anticipate all the specific challenges future specialists will encounter that require a Background in microbiology. However, they must be fully equipped to assess the microbiological safety of both food and non-food products—recognizing it as a vital consumer attribute in today's market and a key competitive advantage globally. They must also understand the legal and ethical responsibility associated with trading substandard or hazardous goods, and possess the skills to organize and conduct microbiological safety audits.

Class="center">Appendix 1. SAFETY GUIDELINES IN THE MICROBIOLOGY LABORATORY

1. Working with live pathogens is strictly prohibited in educational microbiology laboratories.

2. Workspaces used for handling microbial cultures must be disinfected both before and after work using lysol or chloramine solutions, or 70% (v/v) isopropyl or ethyl alcohol.

3. Workspaces must be kept free of clutter and unnecessary items.

4. All containers holding Reagents and other substances must be properly labeled or numbered to prevent mix-ups.

5. Never taste chemical substances or nutrient media.

6. Laboratory work without protective clothing is prohibited. Personnel must wear a white lab coat at all times inside the microbiology laboratory.

7. Before leaving the laboratory, the duty officer and supervisor must ensure that all gas and water Valves are shut, alcohol burners are extinguished, electrical heating appliances and ventilation systems are turned off, and flammable Materials are safely stored.

8. Vacuum operations must be performed wearing safety goggles, and glassware must be shielded with protective screens or wrapped in a towel.

9. Any heating Procedures involving acids or alkalis must be performed wearing safety goggles.

10. Clean up and organize your workstation upon completing your work.

11. Never leave reagents, caustic alkalis, or acids uncovered.

12. Place used Laboratory glassware in the washing area or wash it yourself.

13. Entering small rooms (inoculation chambers or booths) while the germicidal UV lamp is on is strictly prohibited.

14. Smoking, storing or consuming food and beverages, and chewing gum are strictly forbidden in the laboratory.

15. Only trained and authorized personnel are permitted to operate autoclaves and other pressure vessels!

APPENDIX 2. Classification OF HUMAN PATHOGENIC MICROORGANISMS OF HAZARD GROUPS III AND IV (EXCERPT FROM APPENDIX 5.1 TO SP 1.2.011-94)

Bacteria – Group III

1. Bordetella pertussis

2. Borrelia recurrentis

3. Campylobacter fetys

4. Campylobacter jejuni

5. Clostridium botulinium

6. Clostridium tetani

7. Corynebacterium diphteriae

8. Erysipelothrix rhusiopathiae

9. Helicobacter pylori

10. Leptospira interrogans

11. Listeria monocytogenes

12. Mycobacterium leprae

13. Mycobacterium tuberculosis Mycobacterium bovis Mycobacterium avium of pertussis, relapsing fever, abscesses, septicemia, enteritis, cholecystitis, botulism, tetanus, diphtheria, erysipeloid, gastritis, gastric and duodenal ulcers, leptospirosis, Listeriosis, leprosy, tuberculosis

14. Neisseria gonorrhoeae

15. Neisseria meningitidis

16. Nocarolia asteroides

17. Proactinomyces israelii

18. Salmonella paratyphi A

19. Salmonella paratyphi B

20. Salmonella typhi

21. Shigellaspp.

22. Treponema pallidum

23. Yercinia pseudotuberculosis

24. Vibrio cholerae 01

Group IV

1. Aerobacter aerogenes

2. Bacillus cereus

3. Bacteroides spp.

4. Borrelia spp.

5. Bordetella bronchiseptica Bordetella parapertussis

6. Campylobacter spp.

7. Citrobacter

8. Clostridium perfringens Clostridium novyi Clostridium septicum Clostridium histolyticum Clostridium bifermentans

9. E. coli

10. Eubacterium endocarditidis

11. Eubacterium lentum Eubacterium ventricosum Gonorrhea, meningitis, nocardiosis, actinomycosis paratyphoid A, paratyphoid B, typhoid fever, dysentery, Syphilis, pseudotuberculosis, non-toxigenic diarrhea, enteritis, foodborne toxicoinfection, lung abscesses, bacteremia, tick-borne spirochetosis, bronchosepticosis, parapertussis, gastroenteritis, gingivitis, periodontitis local inflammatory processes, foodborne toxicoinfection, gas gangrene, septic enteritis, endocarditis secondary septicemia abscesses

12. Flavobacterium meningosepticum

13. Haemophilus influenzae

14. Hafnia alvei

15. Klebsiella ozaenae

16. Klebsiella pneumoniae

17. Klebsiella rhinoscleromatis

18. Mycobacterium spp. Photochromogens Scotochromogens Nonphotochromogens Rapid growers

19. Mycoplasma hominis 1 Mycoplasma hominis 2 Mycoplasma pneumoniae

20. Propionibacterium avidum

21. Proteus spp.

22. Pseudomonas aeruginosa

23. Salmonella spp.

24. Serratia marcescens

25. Staphylococcus spp.

26. Streptococcus spp. meningitis, septicaemia, Pneumonia, laryngitis, cholecystitis, cystitis, ozaena, pneumonia, rhinoscleroma, microbacterioses, local inflammatory processes, pneumonia, Sepsis, abscesses, foodborne toxicoinfection, local inflammatory processes, sepsis, salmonellosis, local inflammatory processes, sepsis, foodborne toxicoinfection, septicaemia, pneumonia, pneumonia, tonsillitis, polyarthritis, septicaemia, enteritis, colitis, actinomycosis

27. Yersinia enterocolitica;

28. Actinomyces albus

APPENDIX 3. PREPARATION OF CERTAIN STAINS AND CULTURE MEDIA

1. Stains

Basic fuchsin alcoholic solution - saturated

Basic fuchsin, crystalline — 10 g;

Ethyl alcohol, 96%   — 100 mL.

Dissolve the fuchsin in 100 mL of 96% ethanol. The solution can be stored for a long time in a dark Glass bottle with a ground-glass stopper.

Carbol fuchsin - Ziehl's fuchsin

Basic fuchsin, crystalline - 1 g;

Carbolic acid (phenol)  — 5 g;

Ethyl alcohol, 96%   — 10 mL;

Distilled water  — 100 mL.

To prepare the solution, pre-weigh 1 g of crystalline basic fuchsin, place it in a porcelain mortar, and triturate with 5 g of carbolic acid, adding the alcohol in small portions and distilled water until the crystals are completely dissolved; then add the remaining water. Place the prepared stain in an incubator at 37 °C for two days. Afterwards, filter through a fluted paper filter.

Carbol fuchsin is stable and can be stored for a long time. It should be stored in a dark glass bottle with a ground-glass stopper.

Carbol fuchsin can also be prepared by an alternative method:

First, prepare two separate solutions.

Solution 1

Basic fuchsin, crystalline — 1 g;

Ethyl alcohol, 96%   — 10 mL.

Triturate the mixture in a porcelain mortar until the fuchsin crystals are completely dissolved.

Solution 2

Carbolic acid - 5 g;

Distilled water - 95 mL.

The water is heated to 50 °C to facilitate the dissolution of carbolic acid. The second solution is poured into the first, mixed thoroughly, and filtered through a fluted paper filter.

Basic Fuchsin, Aqueous (Pfeiffer's Fuchsin)

Ziehl's carbol fuchsin  — 10 ml;

Distilled water   — 90 ml.

Aqueous fuchsin is unstable and is best prepared immediately before use. The aqueous fuchsin solution can be stored for no more than 10 days.

Carbol Gentian Violet

Gentian violet (crystals) — 1 g;

Ethyl alcohol, 96%   — 10 ml;

Carbolic acid    — 5 g;

Distilled water   — 100 ml.

The preparation technique for carbol gentian violet is the same as that for Ziehl's carbol fuchsin.

When preparing carbol gentian violet, one can also prepare two separate solutions beforehand.

Solution 1

Gentian violet  — 1 g;

Ethyl alcohol, 96%   — 10 ml.

Solution 2

Carbolic acid - 5 g;

Distilled water - 95 ml.

The sequence and method of preparing the First and Second solutions are identical to those for Ziehl's carbol fuchsin.

Preparation of Gentian Violet Staining Papers (according to Sinev)

To prepare the papers, filter paper is first tested for suitability. For this purpose, a strip of filter paper is immersed in an alcoholic solution of the dye (carbol gentian violet) and air-dried. Good quality paper stains evenly without spots. If a small piece of such paper is placed on a glass slide with a few drops of water, it becomes saturated immediately, and the dye passes into solution within a few seconds. The paper to be impregnated is cut into strips (2.5–3 cm wide and 30 cm long) and immersed in the dye so that both surfaces are wetted. The strips are then removed and air-dried at room Temperature or in an incubator at 37 °C.

Staining papers can also be impregnated with an alcoholic gentian violet solution of the following composition:

Gentian violet (crystals) — 1 g;

Ethyl alcohol, 96%   — 100 ml;

Glycerol    — 5 ml.

Methylene blue (saturated alcoholic solution)

Dissolve 3 g of methylene blue in 100 ml of 96% ethanol. Let the solution stand for 2–3 days, shaking it occasionally, and then filter through filter paper. The solution is stable.

Brilliant green solution (malachite green solution)

A saturated aqueous solution is prepared by dissolving 10 g of malachite green in 10 ml of distilled water. Alcohol-water solution: mix 10 g of malachite green with 80 ml of distilled water and 20 ml of 96% ethanol.

Methylene blue 1:40

Mix 1 ml of the saturated alcoholic solution of methylene blue with 40 ml of distilled water.

Methylene blue, alkaline solution (Loeffler's)

To 100 ml of distilled water, add 30 ml of saturated alcoholic methylene blue solution and 1 ml of a 1% aqueous potassium hydroxide solution.

Methylene blue according to Fink

Weigh out separately 0.9 g of disodium hydrogen phosphate, 13.6 g of potassium dihydrogen phosphate, and 0.1 g of methylene blue. Dissolve each portion in 500 ml of distilled water. Mix 0.25 ml of the first solution with 99.75 ml of the second and 100 ml of the third (pH 4.6).

Safranin (aqueous)

Mix 10 ml of a 2.5% safranin solution in 96% ethanol with 100 ml of distilled water.

Lugol's iodine (Gram's modification)

Place 1 g of iodine crystals and 2 g of potassium iodide into a 30–50 ml mortar, grind the mixture with a pestle, add 1 ml of distilled water, continue grinding the crystals, and add another 5 ml of water until the iodine dissolves in the potassium iodide. Quantitatively transfer the solution to a bottle and bring the total volume to 300 ml. The solution is stable for up to 30 days and should be stored in a cool, dark place, preferably in an amber glass bottle.

Lugol's solution for detecting Glycogen and granulose

Iodine crystals   — 1 g;

Potassium iodide    — 3 g;

Distilled water   — 300 ml.

Prepare the solution in the same manner as the previous one.

A strong iodine solution can also be used to detect glycogen

Iodine crystals   — 7 g;

Potassium iodide    — 20 g;

Distilled water   — 100 ml.

Muromtsev's stain

Prepare two solutions:

Solution 1

Basic fuchsin    — 0.15 g;

Alcohol, 96%    — 20 mL;

Crystalline carbolic acid - 10 g.

Solution 2

Methylene blue - 2.5 g;

Distilled water - 200 mL.

Both solutions are mixed and filtered through filter paper.

India Ink Solution for Capsule Staining

Mix 10 mL of liquid natural India ink with 90 mL of distilled water. Then, centrifuge the solution for 15–20 min. Transfer the upper layer to a test tube and autoclave for 30 min (0.05 MPa, temperature 110 °C). After autoclaving, let the solution stand for two weeks, after which it is ready for use.

2. Culture Media

2.1. General-Purpose Culture Media

Meat-peptone broth. Prepare meat infusion as follows: 1 kg of beef, trimmed of bones, fat, and tendons, is passed through a meat grinder and mixed with 2 L of tap water. The minced meat is steeped in water for 12–24 hours in a cold place. During this time, water-soluble proteins, Amino Acids, Vitamins, CARBOHYDRATES, minerals, and other substances are extracted from the meat. The infusion is filtered through a double layer of gauze, the meat is thoroughly squeezed out, and the filtrate is boiled for 30 min to coagulate the proteins. After removing the fat, the cooled liquid is passed through a cotton-gauze filter and made up to the original volume with water. The meat infusion is dispensed into flasks or bottles and sterilized in an autoclave at 0.1 MPa for 20 min.

To prepare meat-peptone broth, add 1% peptone and 0.5% chemically pure sodium chloride to the meat infusion, boil for 10 min, filter through a fluted filter paper, adjust the pH to 7.2–7.4 with a 10% sodium bicarbonate solution, and boil again for 10 min. Meat-peptone broth should have a straw-yellow color and be perfectly clear. It is dispensed into test tubes and flasks, plugged with cotton-gauze stoppers, and sterilized at a pressure of 0.1 MPa for 20–30 min.

Nutrient broth can also be prepared from meat substitutes (meat extract, fish hydrolysate) According to the instructions on the label.

Meat-peptone Agar is prepared from meat-peptone broth by adding 2–3% minced or powdered agar-agar. The solution is heated to a boil and simmered until the agar is completely dissolved. The solution is then cooled to 50 °C and clarified with whipped egg white (1 egg white per 1 L of medium) mixed with 30 mL of water, brought back to a boil, and simmered for 20 min. The protein coagulates, settles, and clarifies the medium. The hot agar is filtered through a cotton-gauze layer, adjusted to pH 7.2–7.4, dispensed into test tubes or flasks, and sterilized for 20 min at a pressure of 0.1 MPa.

Peptone water. Dissolve 30 g of peptone in 1 L of distilled water and sterilize for 20 min at a pressure of 0.1 MPa.

Peptone water can also be prepared as follows: add 10 g of peptone, 5 g of sodium chloride, and 0.1 g of potassium nitrate to 1 L of distilled water with heating. Filter through filter paper and adjust the pH to 7.6–7.8. Dispense into test tubes and sterilize with flowing steam fractionally for 30 min on each of 3 consecutive days.

Malt wort. Pour 1 L of tap water over 250–300 g of coarsely ground dry barley malt, heat to 48–50 °C, and maintain the temperature for 30 min with constant stirring to prevent lump formation. Then raise the temperature to 55–58 °C, after 30 min to 63 °C, and hold the mixture at this temperature until the starch is completely saccharified. Press the finished medium through a cloth filter to remove the spent grain, then filter through a fluted filter paper. Determine the dry matter (DM) concentration of the filtrate at 20 °C, which is typically 18–20%. Dilute the filtrate with tap water to the desired concentration. For Yeast cultivation, the malt wort should have a concentration of 6–8% DM; for lactic acid bacteria, 8–12% DM; and for Molds, 3–4%, with a medium pH of 5.6–6.0. If the pH is lower, alkalize the wort with a 10% sodium bicarbonate or sodium hydroxide solution. Next, dispense the wort into flasks or test tubes, plug them with cotton-gauze stoppers, and sterilize at 0.05 MPa for 30 min.

Unhopped commercial brewer's wort, adjusted to a specific dry matter content and pH, can also be used to prepare this medium.

Wort agar. When preparing wort agar, add 2% agar to the malt wort. For acid-producing microorganisms, a small amount of chalk is also added. Sterilize the medium for 30 min at a pressure of 0.05 MPa or fractionally with flowing steam.

The medium is used for the isolation, cultivation, and maintenance of Yeasts, molds, and lactic and acetic acid bacteria.

Yeast autolysate. Method 1: Place a homogeneous mass of 1 kg of compressed baker's yeast and 1 L of boiled tap water in an incubator at 50 °C, add a few drops of toluene, and incubate with periodic stirring for 72 hours. Upon completion of yeast autolysis, heat the mass in an autoclave at 0.02 MPa for 30 min. Filter the cooled mass through a double fluted filter paper until completely clear. The clear filtrate contains 0.9% nitrogen. Neutralize the filtrate to pH 6.8–7.0, dispense into test tubes or flasks, and sterilize at a pressure of 0.01–0.05 MPa for 10–20 min.

Method 2: Mix 1 kg of compressed yeast with 4 L of tap water and incubate in an incubator at 55 ⁰C for 24 hours. Then filter the autolysate and sterilize it at 0.05 MPa for 20 min.

2.2. Special (Selective) Culture Media

2.2.1. Media for the Detection of Staphylococci

Milk-salt agar: dissolve 6.5 g of sodium chloride in 100 cm³ of nutrient agar by boiling, and sterilize at 0.1 MPa for 20 min. To the melted agar cooled to 45 °C, add 10 cm³ of skimmed sterilized milk per 100 cm³ of agar, mix thoroughly, and pour in a thin layer into Petri dishes. Count the colonies exhibiting a clear halo.

Yolk-salt agar: aseptically add 50 cm³ of egg-yolk solution (1 chicken egg yolk dissolved in 150–200 cm³ of physiological saline) to 150 cm³ of melted and 45 °C-cooled 6% salt agar. Mix rapidly and pour into Petri dishes.

2.2.2. Media for the Cultivation of Yeasts and Microscopic Fungi

Sabouraud medium. Yeast water serves as The basis of this medium. To prepare yeast water, boil 70–100 g of fresh compressed yeast (or 7–10 g of dry yeast) in 1 L of distilled water for 20–30 min and let it settle in a tall cylinder in a cold place for 12 hours. Decant the supernatant, add another 1 L of water, boil for 30 min, filter, and adjust the pH to the required value. Sterilize the prepared medium fractionally for 20 min over 2–3 days. To 100 mL of sterile yeast water, add 1% peptone and 2% agar; after the agar dissolves, incorporate a 4% glucose or maltose solution, filter, dispense into test tubes, and sterilize at 0.05 MPa for 20 min.

The medium may also be prepared using standard 1% peptone water.

Rieder's synthetic medium for yeasts. The medium composition (in g/L) is as follows: ammonium sulfate 3, magnesium sulfate 0.7, calcium nitrate 0.04, sodium chloride 0.5, potassium dihydrogen phosphate 1.0, and dipotassium hydrogen phosphate. The initial pH of the medium is 6.6. To study yeast reproduction, add 2% sugar; for Fermentation studies, add 5–10%. The complete synthetic medium also contains vitamins (in mcg/mL): Inositol 5, biotin 0.0001, pantothenic acid 0.25, thiamine 1.0, pyridoxine 0.25, and nicotinic acid 0.5. Sterilize the medium in an autoclave at a pressure of 0.1 MPa.

Potato-glucose agar. Peel and slice 200 g of potatoes, pour 1 L of distilled water over them, and boil for 1 hour. Filter the broth, add water back to the filtrate to restore the initial volume, and incorporate 2% glucose and 2–3% agar-agar. Dispense the medium into test tubes or flasks and sterilize at 0.1 MPa for 10 min. Prior to use, adjust the pH to 3.5 using a 10% sterile solution of anhydrous citric acid.

Czapek's synthetic medium for fungi. Medium composition (in g/L): sucrose or glucose 30, potassium dihydrogen phosphate 1.0, sodium nitrate 2.0, magnesium sulfate 0.5, potassium chloride 0.05, iron sulfate 0.1, and agar 20. Leach the weighed agar and add it to the specified ingredients previously dissolved in 1 L of distilled water. Heat with flowing steam, and adjust the pH to 4.0–5.5 using a 10% solution of citric acid or sodium hydroxide. Filter, dispense into test tubes, and sterilize fractionally with flowing steam for 3 days, 30 min each day.

Complete Lysine medium for the detection of imperfect yeasts. Add the following ingredients (in g/L) to 1 L of tap water: glucose 50, magnesium sulfate 1, potassium dihydrogen phosphate 2, potassium lactate 12 mL of a 50% solution, L(+) lysine monohydrate 1, and vitamin solution (per 100 mL of sterile distilled water, add (in g) inositol 2, calcium pantothenate 0.4, nicotinamide 0.5, and thiamine hydrochloride 0.1), along with 20 g of agar. The pH of the medium is 5.0–5.2. Dispense the medium into test tubes and sterilize for 15 min at 0.1 MPa.

Acetate medium for the detection of imperfect yeasts. For 1 L of tap water, take 10 g of sodium acetate, 10 g of ammonium chloride, 5 g of glucose, and 3 mL of yeast autolysate; dispense into test tubes and sterilize at a pressure of 0.05 MPa for 30 min.

2.2.3. Media for the Cultivation of Lactic Acid Bacteria

Pour whole milk into test tubes or flasks and sterilize at 0.1 MPa for 10 min. This medium is used to study the physiological properties of lactic acid bacteria.

Skimmed milk is separated from cream by centrifugation, dispensed into test tubes or flasks, and sterilized under the same conditions as whole milk. This medium is used to study the physiological properties of microbes and for group quantitative enumeration of lactic acid bacteria.

Hydrolyzed milk. Adjust the pH of sterile skimmed milk to 7.6–7.8. Heat the milk to 45 °C and add 0.5–1 g of pancreatin (previously dissolved in warm water) and 5 mL of chloroform per 1 L of milk. Tightly close the milk container with a cork stopper, mix the mixture thoroughly, and place it in an incubator at 40 °C for 18–24 hours. Then, decant the resulting clear liquid and filter it through filter paper. Dilute the filtrate twofold with water, adjust the pH to 7.0–7.2, and sterilize at 0.1 MPa for 15 min.

Hydrolyzed milk agar. Incorporate 1.5–2.0% agar-agar into hydrolyzed milk. Heat the mixture to a boil and maintain until the agar is completely dissolved. Filter the hot medium through a cotton filter, dispense into test tubes or flasks, and sterilize at 0.1 MPa pressure for 10–15 min.

2.2.4 Medium for the Quantitative Enumeration of Putrefactive Bacteria

Milk agar is prepared by adding 20% hot sterile skimmed milk to a sterile melted 2% aqueous agar-agar solution. This medium is used for the quantitative enumeration of proteolytic and peptonizing bacteria (micrococci, mammococci). Zones of proteolysis and peptonization form around the colonies of putrefactive bacteria.

Beef fat medium. The COMPOSITION OF THE medium includes: peptone 1 g, yeast autolysate 0.3 g, disodium phosphate 0.1 g, agar 1.5 g, and distilled water up to 100 mL; pH 7.0–7.4.

Prepare sterile beef fat separately in test tubes. Sterilize the medium at 121 °C (0.1 MPa) for 15 min.

2.2.5. Media for the Detection and Identification of Coliform Bacteria

Kessler medium: add 10 g of peptone and 50 mL of fresh bovine Bile to 1 L of tap water. Boil the mixture on a water bath for 30 min with stirring, then filter through cotton wool, add 2.5 g of glucose, and bring the volume to 1 L. Adjust the reaction of the medium (pH 7.4–7.6) and add 2 mL of a 1% aqueous crystal violet solution. Dispense the medium into test tubes or flasks equipped with fermentation tubes in volumes required for testing individual products. Sterilize at 0.05 MPa for 20 min. The color of the medium should be dark violet.

Lactose-peptone (glucose-peptone) medium. Dissolve 10 g of peptone, 5 g of sodium chloride, and 5 g of lactose (glucose) in 1 L of distilled water with heating. After the ingredients dissolve, adjust the pH to 7.4–7.6. Dispense the medium into test tubes and sterilize at 0.05 MPa for 10–15 min.

Semi-solid medium with lactose or mannitol (glucose). Dissolve 10 g of peptone, 5 g of sodium chloride, and 4–5 g of agar-agar in 1 L of distilled water, bring to a boil, adjust the pH to 7.2–7.4, and add 1 mL of a 1.6% alcoholic bromothymol blue solution. Sterilize at 0.1 MPa pressure for 20 min. Incorporate 5 g of lactose or mannitol (glucose) into the melted medium, heat to a boil, dispense into sterile test tubes to a height of 3–5 cm, and sterilize at 0.05 MPa for 10–15 min. A properly prepared medium has a green color with a bluish tinge (bottle-glass color). The shelf life of such a medium does not exceed 2 weeks.

Endo medium (fuchsin-sulfite agar): dissolve 1 g of lactose in 5 mL of sterile water, heat on a water bath at 100 °C for 5 min under sterile conditions, and add to 100 mL of melted 2% meat-peptone agar with a pH of 7.6–7.8. In a separate sterile test tube, place 0.5 mL of freshly prepared and filtered 10% basic fuchsin solution, to which a freshly prepared 10% sodium sulfite solution is added until a pale pink color is obtained. Incorporate the resulting mixture into the melted lactose agar, mix thoroughly while avoiding foaming, and pour into sterile Petri dishes. Endo medium must be freshly prepared.

2.2.6. Media for the Detection of Sulfite-Reducing Clostridia and Other Anaerobes

Iron-sulfite agar. The basic medium. Add 10 g of glucose to 1 L of sterile molten nutrient agar, heat until dissolved, dispense metrically into flasks, and autoclave at 0.05 MPa for 10-15 min.

A 20% sodium sulfite solution and an 8% iron(II) sulfate (ferrous sulfate) solution are prepared immediately before use in sterile glassware using sterile distilled water. The sodium sulfite solution is heated until completely dissolved. Before performing the analysis, add 5 mL of the 20% sodium sulfite solution to 100 mL of the molten basic medium, mix well, then add 1 mL of the 8% sodium sulfite solution, mix again, and dispense into sterile tubes or flasks following standard sterility procedures.

Thioglycollate broth (fluid thioglycollate medium / Kitt-Tarozzi medium). Pre-boiled and water-washed pieces of Liver or meat are placed into test tubes and filled with meat-peptone broth containing 1% glucose up to 4/5 of the tube volume. A 1-cm-high layer of liquid paraffin is poured on top. The medium is sterilized at 0.1 MPa for 15 min.

APPENDIX 4. PREPARATION OF DISINFECTANT SOLUTIONS

1. Preparation of Chlorine-Based Disinfectants

1. Bleaching powder (chloride of lime) exhibits strong bactericidal activity. In microbiological practice, it is used as a powder, a 10–20% milk of lime suspension, and a 0.2–10% clarified solution.

2. Procedure for preparing milk of lime.

Mix 1 kg of dry bleaching powder with 10 L of water to form a “milk of lime” suspension, and leave it in a dedicated room in a dark container for 24 hours. The resulting 10% clarified bleaching powder solution is then decanted into a suitable dark glass container, labeled with the preparation date, and stored in a dark place, as active chlorine degrades quite rapidly upon exposure to light.

3. Chloramine contains 25–26.5% active chlorine.

Microbiological laboratories use 0.2–10% chloramine solutions. The chloramine solution is prepared immediately prior to use. To prepare the solutions, the required amount of chloramine is dissolved in water preheated to 50–60 °C.

For the preparation of:

✵ a 0.5% solution, use 5 g of chloramine per 1 L of water;

✵ a 1% solution, use 10 g of chloramine per 1 L of water;

✵ a 3% solution, use 30 g of chloramine per 1 L of water;

✵ a 5% solution, use 50 g of chloramine per 1 L of water.

First, add the required amount of chloramine to a small volume of water, mix thoroughly, and then bring it up to the required final volume.

2. Preparation of Stock Solution of Bleaching Powder

1. Take a suitable container (dark glass bottles with ground-glass stoppers, enameled, or glassware) and place 1 kg of dry bleaching powder into it. While stirring continuously, gradually add a small amount of water until a slurry is formed. Then, continuing to stir, add water up to a total volume of 10 L. That is, to prepare a 10% bleaching powder solution, use: 1 kg of dry bleaching powder + 9 L of water = 10 L of solution.

2. Leave the freshly prepared solutions to stand for 24 hours in a cool, dark room in a closed container. After 24 hours, carefully decant the clarified solution without disturbing the sediment into another container (dark glass, enameled, or plasticware) for storage for up to 10 days.

3. When preparing the stock solution, the container (its attached label) must indicate the preparation date, solution concentration, position, and full name of the person who prepared it.

Caution! The Use of galvanized cookware or containers is strictly prohibited.

3. Phenol-Based Disinfectants

1. Carbolic acid. Liquid carbolic acid contains 90% crystalline phenol and 10% water. Microbiological laboratories use 3–5% carbolic acid solutions. The activity of phenol increases when it is dissolved in hot water (40–50 °C).

Procedure for preparing carbolic acid solutions for microbiological research:

To prepare a 3% carbolic acid solution, dissolve 30 g of crystalline phenol or 33 ml of liquid carbolic acid in 1 liter of water.

To prepare a 5% carbolic acid solution, dissolve 50 g of crystalline phenol or 55 ml of liquid carbolic acid in 1 liter of water.

Crystalline phenol or liquid carbolic acid can cause Skin irritation upon contact, and severe Burns at high concentrations. Therefore, carbolic acid must be handled with extreme care. Rubber gloves should be worn when preparing solutions. If carbolic acid gets on the skin, immediately wash it off with warm soapy water or 40° ethyl alcohol.

2. Chromic mixture. Preparation procedure:

Pour 150 ml of concentrated sulfuric acid into a flask and add 25 g of potassium dichromate. Leave the mixture to stand until fully dissolved. After 24 hours, the dark orange solution is ready for washing glassware. Before use, the mixture should be heated to 45–50 °C. If the color of the mixture changes to dark green, it indicates that it is unfit for use.

APPENDIX 5. REGULATED MICROBIOLOGICAL PARAMETERS OF FOOD PRODUCTS (EXTRACT FROM SanPiN 2.3.2. 1078-01)

1. Meat and meat products: poultry, eggs, and processed egg products

2. Milk and dairy products

3. Confectionery

4. Oilseeds and fat products

5. Beverages

6. Infant foods (dairy-based products)